Abstract HIV is estimated to produce 1-10 billion new viral particles per day in an infected individual. This high rate of viral replication, paired with an inability of the viral reverse transcriptase to correct misincorporated nucleotides, generates mutations that confer resistance to antiretroviral drugs. While suppression of HIV replication was achieved in 1995 by combining three drugs with different resistance mechanisms, drug-resistant viruses are readily selected when intracellular levels of two of the three drugs wane. Not long after the advent of successful antiretroviral treatment (ART), rapid treatment failure was noted to occur in individuals who acquired drug-resistant viruses. To avoid this outcome, testing for HIV-drug-resistance prior to initiation of ART became routine. HIV sequence analysis, which costs >$500/specimen, is preferred for testing. However, in the resource-poor communities, this cost is prohibitive. As transmitted drug resistance is increasing in Africa, an economical drug resistance test is needed to prevent drug-resistant viruses from undermining the health gains and reduced HIV transmission rates that have resulted from ART programs. We have adapted an inexpensive point mutation assay to detect mutations conferring HIV-drug-resistance to 1st-line ART for use in resource-poor communities. The assay amplifies the HIV polymerase gene, ligates oligonucleotide probes to specifically detect point mutations that confer drug resistance, and ligation products are detected in an EIA plate format. In research studies, this oligonucleotide ligation assay (OLA) has predicted virologic failure of 1st-line ART among Thais and Kenyans. OLA could be effective in patient management, but the complex laboratory procedures have been an obstacle to adoption in low-resource laboratories. Here, we propose to re-engineer the OLA so that it is rapid and simple to perform. Our goal is to systematically simplify the OLA procedure to reduce the assay time from 8+ hours to about an hour and make it accessible to minimally-trained laboratory personnel. We have assembled a team with the expertise needed to simplify OLA through the following modifications: Concentrate nucleic acids from blood specimens using stimuli-responsive reagents. Amplify HIV DNA by isothermal strand displacement amplification. Develop a one-pot ligation that targets the HIV codons most relevant to 1st-line ART. Develop a simple and rapid paper strip test for multiplexed detection of all mutant codons. Combine the re-engineered protocols into a rapid and simplified kit (OLA-Simple). The OLA-Simple kit will advance HIV care in resource-poor areas by allowing rapid implementation of appropriate ART prophylaxis for individuals inadvertently exposed to HIV, and to women in late pregnancy. Recent data suggest that treatment of acute HIV infection may prevent persistent infection, which if confirmed, would present compelling and urgent need for a rapid assay to detect HIV-drug-resistance testing to ensure the appropriate ART.